Biology subjects

Thoma, N. H.

Publications and source records attributed to Thoma, N. H..

4 recordsLinked to original sources

bioRxiv2025

E2F1 induces a G0-G1 reentry transcriptional program without changing chromatin accessibility

Quiescent cells actively repress cell-cycle genes via chromatin-based mechanisms to maintain a non-dividing state, yet remain poised to reenter upon stimulation. E2F1, a canonical activator of cell-cycle genes, is sufficient to induce reentry from quiescence, but how it overcomes chromatin-mediated repression remains unclear. Here, we show that inducible E2F1 expression triggers exit from quiescence and progression through the cycle without changes in chromatin accessibility, by harnessing regulatory elements with limited, pre-existing accessibility. Using time-resolved transcriptomics, we demonstrate that E2F1 induces an accelerated transcriptional program compared to serum. Unlike serum, which triggers broad chromatin remodeling, E2F1-induced activation occurs in a context of limited accessibility. ChIP-seq reveals that E2F1 directly binds target sites in quiescent cells to upregulate canonical genes. Biochemical reconstitution shows that E2F1 binds nucleosomes and accesses internal E2F sites within histone-wrapped DNA. These findings suggest that E2F1 can engage nucleosome-associated DNA and initiate transcription without major chromatin reorganization, redefining transcription factor-chromatin dynamics during cell fate transitions and establishing E2F1 as a potent regulator of cell-cycle reentry.

cell biology↗

bioRxiv2025

Tandem association of CLOCK:BMAL1 complexes on DNA enables recruitment of CBP/p300 through multivalent interactions

The basic helix-loop-helix PER-ARNT-SIM (bHLH-PAS) transcription factor CLOCK:BMAL1 interacts with E-box motifs in the context of nucleosomes to elicit a circadian pattern of gene expression that oscillates with approximately 24 hour periodicity. Core clock genes and other highly rhythmic targets of CLOCK:BMAL1 typically possess a tandem arrangement of E-boxes that is required for robust oscillations. Here, we show that the presence of tandem E-boxes enables CLOCK:BMAL1 to bind more internal sites on the nucleosome, leading to release of DNA from the histone core and the presentation of multiple coactivator binding motifs in close proximity to facilitate multivalent interactions with the coactivator CBP/p300. We show that the transactivation domain (TAD) of BMAL1, essential for CLOCK:BMAL1 activity, interacts with several modular domains of CBP. Deletion of these CBP domains or chemical inhibition of protein-protein interactions with CBP significantly reduces or eliminates CLOCK:BMAL1-driven activity. Altogether, this suggests that multivalent interactions with CBP may play a role in the ability of tandem CLOCK:BMAL1 heterodimers to recruit this limiting cofactor in cells.

biophysics↗

bioRxiv2023

Structural insights into viral hijacking of p53 by E6 and E6AP

The E3-ubiquitin ligase E6AP degrades p53 when complexed with the viral protein E6 from human papilloma virus (HPV), which contributes to the transformation of cells in HPV-related cancers. Previous crystal structures of the E6AP-E6-p53 ternary complex have implicated a peptide containing an LxxLL motif from E6AP as the interface between the three proteins. However, the contributions to the ternary complex from the remainder of the E6AP protein remain unknown. We reexamined this complex using cryo-EM and full-length proteins and find additional protein interaction interfaces involving a previously uncharacterized domain of E6AP. Additionally, we observe that the ternary complex forms both 1:1:1 and 2:2:2 stochiometric complexes comprised of E6AP, E6 and p53.

biophysics↗

bioRxiv2019

Live analysis and reconstruction of single-particle cryo-electron microscopy data with CryoFLARE

Efficient, reproducible and accountable single-particle cryo-electron microscopy structure determination is tedious and often impeded by lack of a standardized procedure for data analysis and processing. To address this issue, we have developed the FMI Live Analysis and Reconstruction Engine (CryoFLARE). CryoFLARE is a modular open-source platform offering easy integration of new processing algorithms developed by the cryo-EM community. It provides a user-friendly interface that allows fast setup of standardized workflows, serving the need of pharmaceutical industry and academia alike who need to optimize throughput of their microscope. To consistently document how data is processed, CryoFLARE contains an integrated reporting facility to create reports. Live analysis and processing parallel to data acquisition are used to monitor and optimize data quality. Problems at the level of the sample preparation (heterogeneity, ice thickness, sparse particles, areas selected for acquisition, etc.) or misalignments of the microscope optics can quickly be detected and rectified before data collection is continued. Interfacing with automated data collection software for retrieval of acquisition metadata reduces user input needed for analysis, and with it minimizes potential sources of errors and workflow inconsistencies. Local and remote export support in Relion-compatible job and data format allows seamless integration into the refinement process. The support for non-linear workflows and fine-grained scheduling for mixed workflows with separate CPU and GPU based calculation steps ensures optimal use of processing hardware. CryoFLAREs flexibility allows it to be used for all types of image acquisitions, ranging from sample screening to high-resolution data collection, and offers a new alternative for setting up image processing workflows. It can be used without modifications of the hardware/software delivered by the microscope supplier. As it is running on a server in parallel to the hardware used for acquisition, it can easily be set up for remote display connections and fast control of the acquisition status.

biophysics↗